Laser-Scatter Measurement Instrument For Organism Detection And Related Network

ABSTRACT

An optical measurement instrument is an integrated instrument that includes an optical cavity with a light source, a sample cuvette, and an optical sensor. The instrument can be used for taking measurements of organism concentration in one or more samples. Preferably, the instrument holds multiple, individually-loaded, independent fluid samples and determines bacteria concentration via a forward-scattering signal. The instrument can incorporate onboard incubation to promote bacterial growth in the samples such that, once a certain bacterial concentration is achieved, the higher concentration sample can be used with a mass spectrometer to identify the type of bacteria. The instrument and mass spectrometer can be a part of a network for medical diagnostic testing data where data is stored in a manner that is inherently untainted by patient identifiable information.

RELATED APPLICATIONS

The present application claims priority to (i) U.S. ProvisionalApplication Ser. No. 62/107,931, filed Jan. 26, 2015, titled“Multi-Sample Laser-Scatter Measurement Instrument With IncubationFeature” (ii) U.S. Provisional Application Ser. No. 62/100,800, filedJan. 7, 2015, titled “System And Method For Detecting and IdentifyingBacteria Type in a Fluid Sample,” (iii) U.S. Provisional ApplicationSer. No. 62/151,065, filed Apr. 22, 2015, titled “Networked BiologicalData Collection System For Use With Laser-Scatter MeasurementInstruments,” and (iv) U.S. application Ser. No. 14/562,304 titled“Cuvette Assembly Having Chambers for Containing Samples to be Evaluatedthrough Optical Measurement,” filed on Dec. 5, 2014, each of which isherein incorporated by reference in entirety.

COPYRIGHT

A portion of the disclosure of this patent document may contain materialwhich is subject to copyright protection. The copyright owner has noobjection to the facsimile reproduction by anyone of the patentdisclosure, as it appears in the Patent and Trademark Office patentfiles or records, but otherwise reserves all copyright rights whatsoever

FIELD OF THE INVENTION

The present invention relates generally to the field of measurements ofbiological liquid samples. Specifically, the present invention relatesto systems and method for determining whether bacteria are present in aliquid sample and, if so, for determining the effect of chemoeffectorson the bacteria within the liquid sample.

BACKGROUND OF THE INVENTION

Many applications in the field of analytical research and clinicaltesting utilize methods for analyzing liquid samples. Among thosemethods are optical measurements that measure absorbance, turbidity,fluorescence/luminescence, and optical scattering measurements. Opticallaser scattering is one of the most sensitive methods, but itsimplementation can be very challenging, especially when analyzingbiological samples in which suspended particles are relativelytransparent in the medium.

One particle that often requires evaluation within a liquid is bacteria.The presence of bacteria is often checked with biological liquids, suchas urine, amniotic, pleural, peritoneal and spinal liquids. In a commonanalytical method, culturing of the bacteria can be time-consuming andinvolves the use of bacterial-growth plates placed within incubators.Normally, laboratory results take may take a day or several days todetermine whether the subject liquid is infected with bacteria and thetype of bacteria.

Quantification of bacteria, yeast, and other organisms in fluid can beuseful for medical diagnosis, drug development, industrial hygiene, foodsafety, and many other fields. Measurement of light scattering andabsorption in samples is a known method for approximating theconcentration of organisms. For example, techniques for detecting andcounting bacteria are generally described in U.S. Pat. Nos. 7,961,311and 8,339,601, both of which are commonly owned and are hereinincorporated by reference in their entireties.

Accordingly, there is a need for an improved systems and methods thatquickly determine whether bacteria is present in the fluid sample anddetermine the effect of chemoeffectors on a fluid sample. There is alsoa need for an improved systems and methods that more quickly determinethe type of bacteria after it is determined that bacteria is present.

Regarding the data collection from medical testing, there are a widevariety of tests conducted in medical labs using collected patientspecimens. These tests, performed “in vitro” can include physical,chemical, and microbiology measurements to determine patient state ofhealth, or to advise a care path. Commonly, these tests are conducted ininstruments or workstations that autonomously generate measurements andinterpreted results. Results are issued by report to a proximate user inthe lab (e.g., a lab operator), and may be collected by one of a varietyof available general-purpose Laboratory Information Systems (“LIS”) thatmanage lab reporting and billing, and can be thereby be viewed by adoctor or other user at a location away from the instrument (a remoteuser). By this method, the lab is able to communicate the interpretedresults from a variety of tests and instruments to users for anyindividual patient as part of the patient care record, to archive theresults in a findable location indexed by the patient identity, and torecord the activities for billing and other purposes.

In these installations, the central database for the LIS does not assistin the interpretation of the data, or impact the algorithms ofinterpretation for the instruments. In operation, a test or instrumentmay typically process the sample in a container or disposable which isdirectly marked, tagged or labeled, or otherwise uniquely affiliatedwith a test event identifier or Accession Number. The test data isgenerally retrieved from the instrument in its fully interpreted form,and is directly indexed to a unique test event identifier, such as thelab Accession Number. This record may also include patient informationsuch as age, gender, specifics of health and care, location, and date.For some combinations and circumstances, this information could becorrelated to create patient identifiable and private data, therebyrequiring the LIS to be designed, operated, and maintained in such a waythat such information retains its security in accordance with privacylaw. Additionally, results from tests are generally indexed to a testidentifier such as lab Accession Number, and recorded in the patientcare record along with other private information, and therefore thisdata can also be considered a potentially a privacy/security concern.

There is also a need for an improved systems and methods to create adata network that can be used to collect and store medical diagnosticdata in a way that is inherently immune to privacy concerns because nosingle database contains both the test data with any private data orcollection of data which could be combined to create private data or beconstrued to constitute Patient Identifiable Information (“PII”). At thesame time, the network includes secure software to retrieve, analyze,and correlate data from individual tests from the various databases tothus momentarily create an interpreted result, indexed to the accessionidentifier, and which can be delivered in digital or printed in hardcopy form for the user or LIS, and which is then deleted from the systemwith no enduring record. In one preferred embodiment, the instrumentsused within the network that create data include the instruments testingfor the presence and concentration of bacteria from laser scattering (orfrom optical instruments measuring the absorbance, turbidity,fluorescence/luminescence, and optical scattering of fluids). The datafrom these instruments is stored in a separate database than a databasehaving any PII. The data may include the results of variouschemoeffectors on a liquid sample containing bacteria.

SUMMARY OF THE INVENTION

The present invention includes several instruments for takingmeasurements of organism concentration in multiple samples as aproduction tool for microbiology. A first instrument holds multiple,individually-loaded, independent fluid samples and determines bacteriaconcentration via a forward-scattering signal. The instrument canincorporate onboard incubation to promote bacterial growth in thesamples during the test.

The instrument is preferably an integrated instrument that includes anoptical cavity with a light source, a sample cuvette, and an opticaldetector. All are enclosed within a light-tight enclosure. The lightsource and sensor/detector are on a bench that is on a translationalmechanical stage such that optical beam can be moved to multiple samplecontainers by mechanical or optical mechanisms and components.

In another embodiment of the first instrument, there is a fixed opticalbeam and the multiple samples can be moved sequentially into the opticalbeam by being translated. Or, the multiple samples can be movedsequentially into the optical beam because the samples are configuredaround a pivot point and can be rotated into a beamline for sequentialmeasurement. In both embodiments, the sample container is preferablyheld in close proximity to a source of heat that is thermostaticallycontrolled to provide incubation warmth to the liquid sample contained.

Alternatively, the present invention is an optical measuring instrumentfor determining a concentration of bacteria in a plurality of fluidsamples. The instrument comprises a housing, a plurality of fluidcontainers, a light source, at least one sensor, and a heating element.The housing has a substantially light-tight enclosure. Each of the fluidcontainers holds a corresponding one of the plurality of fluid samples.Each of the fluid containers has an input window and an output window.The light source within the housing provides an input beam fortransmission into the input windows of the fluid containers and thoughthe corresponding fluid samples. The input beam creates aforward-scatter signal associated with the concentration of bacteria.The at least one sensor within the housing detects the forward-scattersignal exiting from the output windows. The heating element within thehousing maintains the fluid samples at a desired temperature toencourage bacterial growth in the fluid samples over a period of time.At least one of the input beam and the fluid containers are movablerelative to each other so that the input beam sequentially addresseseach of the plurality of fluid samples.

In yet a further aspect, the present invention is a method ofdetermining the concentration of bacteria in a plurality of fluidsamples by use of an optical measuring instrument. The method comprises,within the optical measuring instrument, incubating the fluid sampleswhile each of the fluid samples is within a corresponding one of aplurality of cuvette chambers. Each cuvette chamber has a first windowfor receiving an input beam and a second window for transmitting aforward-scatter signal caused by the input beam. The method furthercomprises during the incubating, repeatedly transmitting the input beamthrough each of the fluid samples and measuring a series offorward-scatter signals for each of the fluid samples, and determiningthat at least one fluid sample includes a concentration of bacteria inresponse to changes in the forward-scatter signals within the series offorward-scatter signals for the at least one fluid sample.

Alternatively, the present invention is an optical measuring instrumentfor determining a concentration of bacteria in a plurality of fluidsamples. The instrument includes a plurality of cuvette assemblieshaving optical chambers for receiving a respective one of the pluralityof liquid samples. Each of the optical chambers includes an entry windowfor allowing transmission of an input light beam through the respectiveliquid sample and an exit window for transmitting an optical signalcaused by the bacteria within the respective liquid sample. Each cuvetteassembly has a first pair of registration structures associatedtherewith. The instrument also includes a platform structure withmultiple second pairs of registration structures for mating with thefirst pair of registration structures of the plurality of cuvetteassemblies. The instrument further includes a light source producing theinput light beam and a sensor for receiving the optical signal caused bythe bacteria.

In yet another aspect, the present invention is an optical measuringinstrument for determining a concentration of bacteria in a plurality offluid samples. The instrument comprises a plurality of cuvetteassemblies having optical chambers for receiving a respective one of theplurality of liquid sample. Each of the optical chambers includes anentry window for allowing transmission of an input light beam throughthe respective liquid sample and an exit window for transmitting anoptical signal caused by the bacteria within the respective liquidsample. The instrument includes a heating system that permits acontrolled incubation of the fluid samples. The instrument also includesa light source for producing the input light beam and a sensor forreceiving the optical signal. The light source being periodicallyoperational during the controlled incubation so as to allow the sensorto receive a series of optical signals that are used for determining theconcentration of bacteria within each of the plurality of fluid samples.

The present invention can also be considered to be an opticalmeasurement system for use in optically measuring bacteria within aliquid sample. The instrument comprises (i) a light source for producingthe input beam, (ii) a sensor for receiving a forward-scatter signalcaused by the input beam passing through a container containing thefluid sample with the bacteria, (iii) a heating system that permits acontrolled incubation temperature for the fluid sample, and (iv) amoveable optical bench. The light source and the sensor are mounted onthe optical bench and the movement of the optical bench permits thefluid sample to be placed into a path of the input beam.

In another aspect, the present invention is a system and method that (i)detects the presence of bacteria in a liquid sample, (ii) determineswhen a certain bacteria concentration is present in the liquid sample,and (iii) in response to a predetermined bacteria concentration beingpresent, identifies the type of bacteria through use of a microbialidentification device. An optical measurement system hasfluid-sample-holding cuvettes (preferably multi-chamber cuvettes) andon-board incubation functionality, such that it can detect the presenceof the bacteria and incubate the fluid sample until the predeterminedbacterial concentration is detected in the fluid sample. The opticalmeasurement system preferably uses cuvettes that receive an input laserbeam through one window and transmit through another window aforward-scatter signal indicative of the bacterial concentration withinthe fluid sample.

In another aspect, the present invention is a method of identifyingbacteria in a fluid sample, comprising (i) placing the fluid sample in acuvette having a first window for receiving an input beam and a secondwindow for transmitting a forward-scatter signal indicative of thepresence or absence of the bacteria in the fluid sample, (ii) incubatingthe fluid sample in the cuvette within an optical-measuring instrumentthat provides the input beam, (iii) passing the input beam through thefluid sample while the cuvette is in the optical-measuring instrument,(iv) analyzing the forward-scatter signal from the fluid sample, (v) inresponse to the forward-scatter signal indicating the presence ofbacteria in the fluid sample, continuing to incubate the fluid samplewithin the optical-measuring instrument to increase the concentration ofthe bacteria within the fluid sample and at least partially identify thetype of bacteria within the fluid sample.

In yet a further aspect, the present invention relates to a network formedical diagnostic testing data where data is stored in a manner that isinherently untainted by patient identifiable information or anycollection of data that might be construed to be private patientinformation. Data from instruments networked within such a system may betransmitted, stored, aggregated, analyzed, and re-interpreted withoutconcern about patient privacy or data security, reducing the burdens ofdatabase and network design, operation, maintenance and use.

In an alternative aspect, the present invention is a method ofidentifying bacteria in a fluid sample, comprising (i) placing the fluidsample in a cuvette having a first window for receiving an input beamand a second window for transmitting a forward-scatter signal indicativeof the presence of the bacteria in the fluid sample, (ii) in response toa first forward-scatter signal indicating the presence of bacteria,incubating the fluid sample in the cuvette to increase the bacteriaconcentration, (iii) in response to a second forward-scatter signalindicating a predetermined concentration of bacteria, removing, from thecuvette, the fluid sample having the increased concentration of bacteriaand (iv) placing at least a portion of the bacteria removed from thecuvette in a mass-spectrometry microbial identification device toidentify the type of bacteria.

Alternatively, the present invention is a network for collecting andusing biological data, comprising a plurality of instruments, a firstdatabase, second database, report-generator software module, and adata-mining software module. The plurality of instruments are at remotelocations and each of the plurality of instruments tests a fluid samplefrom a patient. The first database stores a set of raw test data foreach fluid sample from the plurality of instruments. Each set of rawtest data is stored in a manner that is indexed to a test sample ID. Thefirst database lacks any private patient information. The seconddatabase stores an event record that associates the test sample ID and apatient ID. The report-generator software module accesses informationfrom the first database and the second database to develop a test reportfor each patient. The data-mining software module accesses informationfrom only the first database to determine or predict trends from the rawtest data.

In another aspect, the invention is a method of collecting and usingbiological information from a plurality of instruments at differentlocations. The method comprises (i) testing a plurality of fluid samplesfrom a plurality of patients by use of the plurality of instruments,(ii) storing, in a first database, a set of raw test data for each ofthe plurality of fluid samples, such that each set of raw test data isindexed to a test sample ID, (iii) storing, in a second database, eachof the test sample IDs in a manner that is correlated to a patient ID;(iv) accessing, by use of a report-generator software module,information from both the first database and the second database todevelop a report for the patient; and (v) performing, by use of adata-mining software module, analytics on the sets of raw test datastore in the first database.

In another aspect, the present invention is a network for collecting andusing biological data related to bacteria within fluid samples,comprising a plurality of instruments that are at remote locations. Eachof the plurality of instruments for testing a forward-scatter signalthat is used to determine the presence of bacteria in a fluid samplefrom a patient. The network includes a first database for storing a setof raw test data for each fluid sample from the plurality ofinstruments. Each set of raw test data is stored in a manner that lacksprivate patient information. The network also includes a data-miningsoftware module that accesses information from only the first databaseto determine or predict trends from the raw test data related to atleast one of the group consisting of: (i) a direct comparison ofmultiple antibiotics against a certain infection, (ii) a directcomparison of the same antibiotic at different concentrations against acertain infection, (iii) a direct comparison of a new drug against knowndrugs, (iv) an indication of or a detection of an emergence of one ormore incidents of resistant infection in any healthcare site orgeographic region, (v) an indication of or a detection of a certain typeof bacteria has become or may be becoming resistant to a certainantibiotic, (vi) an indication of or a detection of a certain type ofbacteria in a certain geographical region has become or may be becomingresistant to a certain antibiotic, (vii) an indication of or a detectionof a certain type of bacteria in a certain hospital or care unit hasbecome or may be becoming resistant to a certain antibiotic, (viii) anindication of or a detection of the susceptibility or resistance of aninfection pathogen to an antimicrobial agent, molecule, or combinationor sequence of exposure of antimicrobial agent or molecule with orwithout the active involvement of the proximate healthcare providers orclinical microbiologist.

Additional aspects of the invention will be apparent to those ofordinary skill in the art in view of the detailed description of variousembodiments, which is made with reference to the drawings, a briefdescription of which is provided below.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A illustrates an optical-measuring instrument that is capable ofincubating fluid samples by having a controlled internal heating system.

FIG. 1B illustrates the cuvettes of FIG. 2 being placed and registeredwithin the optical-measuring instrument of FIG. 1A.

FIG. 2 illustrates four multi-chamber cuvettes that receive fluidsamples that are placed in the optical-measuring device of FIGS. 1A and1B.

FIG. 3 illustrates a side view of the optical-measuring instrument ofFIGS. 1A and 1B.

FIG. 4 illustrates a top view of the optical-measuring instrument ofFIGS. 1A and 1B.

FIG. 5 illustrates a system control diagram for the optical-measuringinstrument of FIGS. 1A and 1B.

FIG. 6 is an exploded view of one the multi-chamber cuvettes of FIG. 2that is used with the optical-measuring device of FIGS. 1A and 1B.

FIG. 7 is a cross-sectional view through one chamber of themulti-chamber cuvette of FIG. 2 that is used with the optical-measuringdevice of FIGS. 1A and 1B.

FIG. 8 illustrates the cuvette assembly of FIGS. 2, 6 and 7 registeredon a platform or tray (typically heated) that is movable from the openposition in which the instrument's door is opened for loading to theclosed position in which the instrument's door is closed for sampletesting within the optical measurement instrument of FIGS. 1A and 1B

FIG. 9 illustrates an alternative optical-measuring instrument that iscapable of incubating fluid samples in which the cuvettes form part ofthe light-tight closure of the optical-measuring instrument.

FIG. 10 is an isometric view of optical-measuring instrument with fixedoptical elements and multiple cuvettes that are rotated on a rotatableplatform into the light beam for measuring optical characteristics ofmultiple samples.

FIG. 11 is an image of a display device during operation of instrumentof FIG. 1, which includes bacterial growth curves for seven differentfluid samples over a 240-minute period.

FIG. 12 is another image of a display device during operation of theinstrument of FIG. 1 over a 1300-minute period, wherein the bacterialgrowth curves are for eights samples derived from a single fluid samplebut having different antibiotic concentrations.

FIG. 13 illustrates a system for detecting the presence of bacteria in afluid sample, and then determining the type of bacteria that is presentin the fluid sample.

FIG. 14 illustrates a flow diagram to be used with the devices in FIG.13.

FIG. 15 illustrates a flow diagram involving the process that is used todetect the presence of bacteria in a fluid sample, and then determinethe type of bacteria that is present.

FIG. 16A illustrates Day 1 Test Results showing the incubating andoptical-measuring device of FIG. 1 is measuring the proper concentrationof bacteria within a fluid sample.

FIG. 16B illustrates the MALDI raw-data output for Day 1 (“Dry Target”test) at various bacterial concentrations as measured by theoptical-measuring device of FIG. 1.

FIG. 17A illustrates Day 2 Test Results showing the incubating andoptical-measuring device of FIG. 1 is measuring the proper concentrationof bacteria within a fluid sample.

FIG. 17B illustrates the MALDI raw-data output for Day 2 (“Dry Target”test) at various bacterial concentrations as measured by theoptical-measuring device of FIG. 1.

FIG. 18 illustrates the times at which certain concentrations ofbacteria are detected within a urine sample that has been placed in theoptical measuring device of FIG. 1.

FIG. 19 is a schematic of a network that can be used to collect raw datafrom biological tests, such as testing involving the instruments ofFIGS. 1-18, in which the raw data is maintained in a separate databasethat lacks patient identification information for data-mining analytics.

FIG. 20 is a schematic of an inventory of different test kits that canbe received samples from patients and be used in connection with thetest instruments.

FIG. 21 is a schematic of one test kit from FIG. 20 which includes fourdifferent cuvettes assemblies that is to be used for a particular typeof testing.

FIG. 22 is an exemplary flow diagram of the steps that can be used inconjunction with the network of FIG. 19.

While the invention is susceptible to various modifications andalternative forms, specific embodiments will be shown by way of examplein the drawings and will be described in detail herein. It should beunderstood, however, that the invention is not intended to be limited tothe particular forms disclosed. Rather, the invention is to cover allmodifications, equivalents, and alternatives falling within the spiritand scope of the invention as defined by the appended claims.

DETAILED DESCRIPTION

The drawings will herein be described in detail with the understandingthat the present disclosure is to be considered as an exemplification ofthe principles of the invention and is not intended to limit the broadaspect of the invention to the embodiments illustrated. For purposes ofthe present detailed description, the singular includes the plural andvice versa (unless specifically disclaimed); the words “and” and “or”shall be both conjunctive and disjunctive; the word “all” means “any andall”; the word “any” means “any and all”; and the word “including” means“including without limitation.”

FIG. 1A illustrates an optical measuring device 10 (manufactured by theassignee of the present application as the BacterioScan 216R instrument)that can rapidly detect and quantify the concentration of bacteria in afluid sample. As discussed in more detail below, the instrument 10includes on-board incubation, such that reagents to enhance growth arenot necessarily needed (although they can be used). The instrument 10uses laser-scattering technology to quantify bacteria growth in fluidsample sizes as small as 1 ml. In particular, the instrument 10transmits a laser beam through a fluid sample, and measures the scattersignal caused by the bacteria in the fluid sample, preferably through aforward-scattering measurement technique. The on-board incubationprovides for fluid sample temperatures ranging from room temperature upto 42° C. (or higher). The instrument 10 permits for a range of opticalmeasurement intervals over a period of time (e.g., 1-6 hours) todetermine the growth and concentration of the bacteria within the liquidsamples during incubation. The optical measuring instrument 10 candetect and count bacteria by various techniques that are generallydescribed in U.S. Pat. Nos. 7,961,311 and 8,339,601, both of which arecommonly owned and are herein incorporated by reference in theirentireties.

FIG. 1B illustrates cuvette assemblies 110 being inserted into theoptical measurement instrument 10 of FIG. 1A. To do so, a front door 12on the optical measurement instrument 10 is opened and the cuvetteassemblies 110 are placed on a registration and orientation plate orplatform 210 (See FIG. 8) such that the laser-input window andoutput-signal window of each cuvette (FIGS. 6-7) are substantiallyregistered within the optical measurement instrument 10, permittingperiodic optical measurements to be taken of each sample. As shown, theoptical measurement instrument 10 may include up to four cuvettes 110,such that 16 different samples can be tested periodically through theoptical measurement instrument 10.

The optical measuring instrument 10 includes a display device 14 thatprovides information regarding the tests and/or fluid samples. Forexample, the display device 14 may indicate the testing protocol beingused for the samples (e.g., time and temperature) or provide the currenttemperature within the instrument 10. Preferably, the display device 14also includes an associated touchscreen input (or a different set ofinput buttons can be provided) that allows a user to perform some of thebasic functions of the instrument 10, such as a power on/off function, adoor open/close function, a temperature increase/decrease function, etc.

FIG. 2 illustrates four cuvettes assemblies 110, each of which has fouropenings leading to four different chambers that provide for opticalmeasurement of the fluid samples in the four chambers. The opticalmeasurement is preferably a forward-scattering signal measurement causedby bacteria in the fluid sample. The cuvette assemblies 110 aredescribed in more detail in U.S. Publication No. 2015-0160119, titled“Cuvette Assembly Having Chambers for Containing Samples to be Evaluatedthrough Optical Measurement,” filed on Dec. 5, 2014, which is commonlyowned and is hereby incorporated by reference in its entirety. A briefdescription of the cuvette assembly 110 is provided below with referenceto FIGS. 6-8. The cuvette assemblies 110 can be filled with fluidsamples automatically or manually. As shown, the cuvette assemblies 110are filled through the use of a pipette.

FIGS. 3-4 illustrate more of the details of the internal structures andcomponents of the optical measurement instrument 10. In particular, asshown best in FIG. 4, the cuvettes assemblies 110 are loaded onto amovable platform 210 (show in detail in FIG. 8) when the door 12 isopened. Once loading is complete, the platform 210 moves inwardly intothe instrument 10 and the door 12 is rotated to the closed position,creating a substantially light-tight seal. The door 12 has seals and/orgaskets around it so that the instrument 10 provides a light-tightenclosure to ensure proper signal detection by the sensor 22. As such,the movable platform 210 translates back and forth in the direction ofarrow “A” in FIG. 4. The instrument 10 includes a motor 16, such as amotor that operates a gear (e.g., a worm gear) that is actuated toperform the platform movement and the opening and closing of the door12.

An optical bench 18 is located within the instrument 10. A laser 20 (alight source), which provides an input beam 21, and a sensor 22 arecoupled to the optical bench 18 in a fixed orientation. In oneembodiment, the laser 20 is a visible wavelength collimated laser diode.In another embodiment the laser 20 is a laser beam delivered from anoptical fiber. In yet another embodiment, the laser 20 includes multiplewavelength sources from collimated laser diodes that are combined into asingle co-boresighted beam through one of several possible beamcombining methods. In another example, the light source 20 is anincoherent narrow wavelength source such as an Argon gas incandescentlamp that is transmitted through one or more pinholes to provide a beamof directionality. A stepper motor 24 provides translation movement inthe direction of arrow “B” to the optical bench 18, such that the laser20 and the sensor 22 can move from side to side so as to be registeredin 16 discrete positions that correspond to the 16 samples within thefour cuvettes assemblies 110. At each position, the laser 20 isoperational and its input beam 21 causes a forward-scatter signalassociated with the liquid sample in question. The forward-scattersignal is detected by the sensor 22 and is associated with the bacteriaconcentration. As explained in more detail below with respect tocuvettes assemblies 110, each sample undergoes some type of filteringwithin the cuvette assembly 110 and/or outside the cuvette assembly 110such that unwanted particles are substantially filtered, leaving only(or predominantly only) the bacteria. Due to the incubation featurewithin the instrument 10, the necessary environment around the cuvetteassemblies 110 can be controlled to promote the growth of the bacteria,such that subsequent optical measurements taken by the combination ofthe laser 20 and the sensor 22 results in a stronger forward-scattersignal indicative of increased bacterial concentration. The instrument10 includes internal programming that (i) controls the environmentaround the fluid sample and (ii) dictates the times and/ortimes-intervals between optical measurements to determine whether thebacteria has grown and, if so, how much the concentration of bacteriahas increased. The output of the instrument 10 can be seen on a separatedisplay, as shown in FIGS. 11-12.

In addition to the display 14 located on the instrument 10 (andpreferably the input buttons and/or touchscreen on the instrument 10),the instrument 10 also includes a port 30 (e.g., a USB connection port)for communication with an external device such as a general purposecomputer that would be coupled to the display, such as the one shown inFIGS. 11-12. The instrument 10 can receive instructions from an externaldevice that control the operation of the instrument 10. The instrument10 can also transmit data (e.g., forward-scatter signal data,test-protocol data, cuvette-assembly data derived from a coded label 170as shown in FIG. 6, diagnostic data, etc.) from the port 30. Theinstrument 10 also includes an input power port 32 (e.g., A/C power),which is then converted into a DC power supply 34 for use by the motors,laser, sensors, and displays, etc. One or more printed circuit boards 35provide the various electronics, processors, and memory for operatingthe instrument 10.

FIG. 5 illustrates one embodiment for a control system that is locatedwithin the instrument 10. The instrument 10 includes one or more printedcircuit boards 35 that include at least one processor 50 (and possiblyseveral processors) and at least one memory device 60. The processor 50communicates with the memory device 60, which includes various programsto operate the motor(s), the laser, the sensors, the heating system, thebasic operational functionality, diagnostics, etc. The processor 50 isin communication with the functional components of the instrument 10,such as (1) the optical sensor(s) 22 that sense the forward-scattersignals (or other optical signals, such as fluorescence signals), (2)the laser 20 or other light source that creates the light beam 21 istransmitted into the cuvettes, (3) thermocouple sensors 82 thatdetermine the temperature within the enclosure (or associated with thesurface of the cuvette, (4) the heating system 84, such as Kaptonheaters, IR heaters, etc., which are preferably placed on the platformor tray 210 (FIG. 8) on which the cuvettes reside, (5) the motors 16, 24used for opening the door, moving the platform, and moving the opticalbench, (6) the display(s) 14 on the front of the instrument, (7) anyuser input devices 86 (mechanical buttons or touchscreens), and (8) anaudio alarm 88 to alert the operator of the instrument to a particularcondition or event (e.g., to indicate that one or more samples havereached a certain testing condition, such as a high bacterialconcentration, a certain slope in a bacterial-growth curve has beenachieved, or a certain forward-scatter signal exceeds a certain value).

The processor 50 is also communicating with an external systemsinterface 70, such as interface module, associated with the output port30 on the instrument 10. The primary functions of the processor(s) 50within the instrument 10 are (i) to maintain the enclosure within theinstrument 10 at the appropriate temperature profile (temperature versustime) by use of the thermocouples 82 and heating system 84, (ii) tosequentially actuate the laser 10 so as to provide the necessary inputbeam 21 into the samples within the cuvette assemblies 110, (iii) toreceive and store/transmit the data in the memory device 60 associatedwith the optical (e.g., forward-scatter) signals from the sensor(s) 22,and (iv) to analyze the forward-scatter signals to determine thebacterial concentration. Alternatively, the control system or computermodule that controls the instrument 10 could be partially locatedoutside the instrument 10. For example, a first processor may be locatedwithin the instrument 10 for operating the laser, motors, and heatingsystem, while a second processor outside the instrument 10 handles thedata processing/analysis for the forward-scatter signals received by thesensor 22 to determine bacterial concentration. The test results (e.g.,bacterial concentration indication) and data from the instrument 10 canbe reported on the instrument display 14 and/or transmitted by USB,Ethernet, wifi, Bluetooth, or other communication links from theexternal systems interface 70 within the instrument 10 to externalsystems that conduct further analysis, reporting, archiving, oraggregation with other data (such as the network 600 in FIG. 19).Preferably, as discussed in more detail relative to FIGS. 19-22, acentral database receives test results and data from a plurality ofremotely located instruments 10 such that the test data and results(anonymous data/results) can be used to determine trends usinganalytics, which can then be used to derive better and more robustoperational programs for the instrument 10 (e.g., to decrease time pertest, or decrease the energy of the tests by used lower incubationtemperatures).

Referring to FIGS. 6-7, the cuvette assembly 110 includes four separatecuvettes, each of which includes an optical chamber 112 and aliquid-input chamber 114. The internal and external walls of the lowerportion 113 of the main body of the cuvette assembly 110 define theoptical chamber 112. For example, the first optical chamber 112 ispartially defined by the side external wall, an internal wall, and abottom wall of the lower portion 113, as well as the entry and exitwindows 116, 118. The associated liquid-input chamber 114 is partiallydefined by a side external wall, an internal wall, and a pair of frontand back external walls on the upper portion 115 of the main body of thecuvette assembly 110.

Each of the four entry windows 116 is a part of an entry window assembly117 that is attached to the lower portion 113 of the main body of thecuvette assembly 110. Similarly, each of the four exit windows 118 ispart of an exit window assembly 119 that is attached to the lowerportion of the main body opposite the entry window assembly 117. Inother words, the present invention contemplates a single unitary opticalstructure that provides the transmission of the input beam 21 into allfour respective optical chambers 112, and a single unitary opticalstructure that provides for the exit of the forward-scatter signals fromthe respective optical chambers 112. The lower portion 113 of the mainbody includes structural recesses that mate with the correspondingstructures on the window assemblies 117, 119 for registering them in aproper orientation during assembly of the cuvette assembly 110.

An intermediate partition 130 within the cuvette assembly 110 separatesthe lower portion 113 defining the four optical chambers 112 from theupper portion 115 defining the liquid-input chambers 114. Theintermediate partition 130, which is shown as being part of the lowerportion 113 (although it could be part of the upper portion 115),includes four separate groups of openings that permit the flow of liquidfrom the liquid-input chamber 114 into the associated optical chamber112. The openings can be a variety of shapes that permit the flow of theliquid. As shown, the openings progressively get longer moving from theentry window 116 to the exit window 118 because the shape of the opticalchamber 112 increases in area in the same direction. Additionally, thefilter 132 rests upon the intermediate partition 130, such that the samefilter 132 is used for each of the four regions. When the same filter132 is used for all four regions, the interior walls of the upperportion 115 must provide adequate pressure at the filter 132 to preventcrossing fluid flows through the filter 132 between adjacentliquid-input chambers 112. In a further alternative, no filter 132 ispresent because the intermediate partition 130 includes adequate sizedopenings to provide the necessary filtering of the liquid sample, orbecause the liquid samples are pre-filtered before entering eachliquid-input chamber 114.

To provide the initial introduction of the liquid samples into thecuvette assembly 110, the upper structure 138, which is attached to theupper portion 115 of the main body of the cuvette assembly 110, includesfour openings 140 corresponding to the four liquid-input chambers 114.Four sliding mechanisms 142 are located within four correspondinggrooves 144 on the upper structure 138 and are initially placed in anopened position such that the openings 140 are initially accessible tothe user for introducing the liquid samples. Each of the slidingmechanisms 142 includes a pair of projections 148 that engagecorresponding side channels at the edges of each of the correspondinggrooves 144 to permit the sliding action. Within each groove 144, thereis a latching ramp 146 over which the sliding mechanism 142 is movedwhen transitioning to its closed position. A corresponding latch 147(FIG. 4) on the underside of the sliding mechanism 142 moves over thelatching ramp 146 and creates a locking mechanism when the slidingmechanism 142 has been fully moved to the closed position. The upperstructure 138 of the cuvette assembly 110 also includes a grippinghandle 150 that permits the user to easily grasp the cuvette assembly110 during transport to and from the platform 210 within the instrument10 that incorporates the light source 20 and the sensor 22.

To help seal the cuvette assembly 110 after the liquid samples have beenplaced within the respective liquid-input chambers 114, the periphery ofthe sliding mechanism 142 adjacent to the opening 140 can be configuredto tightly mate with the walls defining the groove 144 (or undercutchannels within the groove 144) to inhibit any leakage around theopening 140 in the upper structure 138. Alternatively, a resilientplug-like structure can be located on the underside of the slidingmechanism 142 that fits within the opening 142 create a seal and inhibitleakage. Or, a gasket can be provided around the opening 140 to providea sealing effect on the underside of the sliding mechanism 142. Thecuvette assemblies 110 provide well sealed containment of the samplesthat reduces evaporation loss.

The upper portion 115 and the lower portion 113 of the main body of thecuvette assembly 110 can be attached to each other through varioustechniques, such as ultrasonic welding, thermal welding, with adhesive,or through interfering snap-fit connections. Similarly, the upperstructure 138 can be attached to the upper portion 115 of the main bodythrough similar techniques. And, the window assemblies 117, 119 can beattached to the lower portion 113 through the same attachmenttechniques. The width dimension of the overall cuvette assembly 110across the four cuvettes is roughly 4 cm. The length dimension of theoverall cuvette assembly 110 (i.e., parallel to the input beam) isapproximately 2 cm. The height dimension of the overall cuvette assembly110 is approximately 2 cm, such that each of the liquid input chambers114 is approximately 1 cm in height and each of the optical chambers 112is approximately 1 cm in height (although the optical chambers 112 havea varying height along the length direction due to their conical shape).In some embodiments, each optical chamber 112 is designed to containapproximately 1.2 to 1.5 cubic centimeters (i.e., approximately 1.2 to1.5 ml) of a fluid sample. Each liquid-input chamber 114 is designed tohold slightly more of the liquid sample (e.g., 1.7 to 2.5 ml), which isthen fed into the corresponding optical chamber 112.

Because each of the cuvette assemblies 110 may be used for differentapplications, the cuvette assembly 110 may use barcodes or RFID tags toidentify the type of test supported by the particular cuvette assembly110, as well as other measurement data to be taken. The instrument 10that includes the light source 20 preferably reads the RFID or barcode,and selects the software program with the memory device 60 to run theappropriate optical measurement tests on the cuvette assembly 110.Accordingly, the cuvette assembly 110 preferably includes anidentification label 170, which may include barcodes and/or quickresponse codes (“QR-code”) that provide the necessary coded informationfor the cuvette assembly 110. Other codes can be used as well.Specifically, when bacteria is a particle being checked within theliquid sample, one of the codes on the label 170 may provide theprotocol for the test (e.g., temperature profile over duration of test,frequency of the optical measurements, duration of test, etc.), and theprocessor 50 executes instructions from the memory 60 (FIG. 5)corresponding to the test protocol. Another one of the codes may beassociated with information on the patient(s) from whom the liquidsamples were taken, which may include some level of encryption to ensurethat patient data is kept confidential. Another code may provide aquality-assurance check of the part number or the serial number for thecuvette assembly 110 to ensure that the cuvette assembly 110 is anauthentic and genuine part, such that improper cuvettes are not tested.The code for the quality-assurance check may also prevent a cuvetteassembly 110 from being tested a second time (perhaps after some type ofcleaning) if it is intended for only single use. Again, the instrument110 preferably includes a device to read the codes associated with thelabel 170 (such as an image sensor, a barcode reader/sensor, or aQR-code reader/sensor). Alternatively, the codes on the label 170 can bescanned as the assemblies 110 are placed into the platform 210 (FIG. 8)such that the necessary information is obtained prior to the door 12being closed.

The cuvette assembly 110 also includes a vent 180 (FIG. 7) that extendsfrom the optical chamber 112 into the upper portion 115 of the main bodythe cuvette assembly 110. The vent 180 includes a chimney-like portionthat extends upwardly from the intermediate partition 130. Thechimney-like portion is then received in a channel in the upper portion115, which extends to an opening 182 leading into the liquid-inputchamber 114 just below the upper structure 138 that defines the upperboundary of the liquid-input chamber 114. Accordingly, the gas (e.g.,air) that is initially present in the optical chamber 112 can be readilydisplaced as the optical chamber 112 receives the filtered liquid samplefrom the liquid-input chamber 114 (via the filter 132). The vent 180 canalso lead to the external environment on the outside of the cuvetteassembly 110.

FIG. 8 illustrates how the cuvettes assemblies 110 are registered withthe optical measurement instrument 10 within a registration platform ortray 210, which is a part of the instrument 10. Each of the cuvettesassemblies 110 includes side registration features 192 that undergo asliding engagement within corresponding vertical grooves 212 on pillarsassociated with the registration platform 210. Additionally, lowerregistration features 194 (FIG. 6) can slide within horizontal grooves214 on an upper surface of the registration platform 210. The horizontalgrooves 214 terminate in openings that receive the lower registrationfeatures 194 (illustrated as projections) on the cuvette assembly 110.Finally, the distance between the lower segments of the front and backwalls of the cuvette assembly 110 corresponds to the width of theregistration platform 210 such that cuvette assembly 110 becomes nestledbetween adjacent pillars with the front and back walls overlying thefront and back edges of the registration platform 210.

As can be seen best in FIGS. 6-7, the lower surface of the lower portion113 of the cuvette assembly 110, which includes the lower registrationfeatures 194, is at angle relative to the upper structure 138 of thecuvette assembly 110 and to the input beam from the light source 20 dueto the conical geometry of the optical chamber 112. Accordingly, theupper surface of the registration platform 210 is angled in an opposingmanner that allows the input beam to be generally horizontal (andgenerally parallel to the upper structure 138 of the cuvette assembly110) when the cuvette assembly 110 is placed on the registrationplatform 210. It should be noted, however, that the cuvette assembly 110can be properly registered on the registration platform 210 with lessthan these three distinct registration features illustrated in FIG. 8.

Once the cuvette assembly 110 is nestled properly on the registrationplatform 210, the door motor 16 is actuated, causing the now-loadedregistration platform 210 to be pulled into the instrument 10 and thedoor 12 to be closed. The light source 20 can then sequentially transmitthe input beam through each of the four optical chambers 112 of eachcuvette assembly 110 and the forward-scatter signal associated with theparticles within each of the liquid samples can be sequentially receivedby the sensor 22. The light source 20 and the sensor 22 on the opticalbench 18 are controllably indexed between positions to receive opticalmeasurements taken in adjacent optical chambers 112. As can be seen inFIG. 8, each platform 210 is capable of receiving four cuvetteassemblies 110, such that optical measurements can be taken from sixteendifferent liquid samples within the four cuvette assemblies 110 nestledon the registration platform 210. Of course, the present inventioncontemplates an instrument 10 that uses more or less than four cuvettesassemblies 110.

According to this first embodiment, the instrument 10 has the opticalbeam 21 along a line from the laser 20 (or other light source such as anLED or lamp) and a light/image sensor 22 such as a camera, imager,calorimeter, thermopile, or solid-state detector array. The liquidsamples are contained in the optical chambers 112 of the cuvetteassemblies 110 between the light source 20 and the sensor 22 with atleast one window so that light can transmit through the sample to thesensor 22. The light source 20 producing the optical beam 21 and thesensor 22 are rigidly mounted to a mechanical optical bench 18 (orplate), and the bench 18 is preferably mounted on rails or othermechanical structures for translational motion (or rotational motion)via a stepper motor 24 (or a motorized threaded stage that moves thebench, or a flexible motor-driven belt) so that it can be movedprecisely relative to the sample in the cuvette 110 so that multiplesamples can be optically measured. Additionally, the bench 18 could betranslated to a diagnostic station 90 with no sample present (far rightposition of the optical bench 18 in FIG. 4) so that it can undergoself-testing or diagnostics in which the sensor 22 confirms performanceof the light source 20, and the light source 20 confirms performance ofthe sensor 22, including provisions of a reticle or other opticaldevices that can be sensed to confirm alignment or optical power levels.

The sample-containing cuvettes 110 and the optical components arecontained in an enclosure within the instrument 10 that excludes mostambient light, which might impact the measurement by the sensor.Alternatively, some portion of the sample cuvette or container couldform a light-tight cover on the instrument, as described below in FIG.9.

In this first embodiment, the sample-containing cuvettes 110 aredisposable containers set on the platform 210 or tray or rail, whichpreferably includes the heating system 84, such as electrical resistanceheaters or pelletier devices and the thermal sensors 82, such as commonthermocouples. The heating system 84 and thermal sensors 82 form part ofthe incubation system that provide for appropriate temperature controlsduring operation of the instrument 10. The electronic control system inFIG. 5 provides for the thermostatic control of the temperature of theplatform 210 and, thus, the contained liquid samples can be warmed orcooled (for example, through fans pulling in cooler air to theenclosure) to a set temperature to influence biological or chemicalbehavior of the liquid samples. Alternatively, the samples (and cuvettes110) could be illuminated by optical or infrared (IR) light sources forheating, and the temperature can be measured or implied by direct orremote sensors.

Furthermore, the platform 210 may be equipped with a vibration-producingmechanism to help agitate the samples in the cuvettes 110. For example,a vibration motor can be coupled to the platform and 210 operatedbetween cycles of the laser operation.

FIG. 9 illustrates an alternative optical-measuring instrument 310 thatis capable of incubating fluid samples in cuvettes 312. However, unlikethe previous embodiments, the cuvettes 312 form part of the light-tightclosure of the optical-measuring instrument 310. In particular, thecuvettes 312 have an upper flange that rest on the exterior surface ofthe instrument 310. The exterior surface includes openings sized toreceive the cuvettes in a certain notation, such that the upper flangerests against the exterior surface. When placed within the opticalmeasuring instrument 310, the entrance and exit windows of the cuvettesare properly aligned with an input laser beam 321 from the laser 320 andthe sensor 322 so as to provide proper registration for measuring theforward-scatter signal associated with the liquid sample. As in previousembodiments, the laser 320 and the sensor 322 would be mounted on anoptical bench 318 that translates within the enclosure of the opticalmeasuring instrument 310 by use of a stepper motor 324. As with theprevious embodiments, the functions of the instrument 310 would becontrolled by one or more processors 350. The optical bench 318 mayinclude other optical components, such as lenses and apertures, toproperly develop the laser beam 321 prior to transmission through theliquid sample in the cuvettes 312. The cuvettes 312 may have internalstructures similar to those of the cuvette assemblies 110 in FIGS. 6-7.

FIG. 10 illustrates another embodiment of an optical measuringinstrument 410 that has one or more input beam lines that are fixed,which is different from the previous embodiments in which the beam linesare translated via the moving optical bench, which includes the laserand sensor. In the embodiment of FIG. 10, multiple sample chambers 412(e.g., cuvettes) are held by a translatable or rotatable platform 413that moves each sample into the light path within the optical measuringinstrument 410. The light is developed by a light source, such as alaser 420 and may reflect off a turning mirror 421 before beingtransmitted through the fluid sample within the sample chamber 412. Asensor 422 receives the optical signal (e.g., a forward-scatter signal),which is then processed/analyzed to determine the presence and/or growthof bacteria over a period of time. The optical measuring instrument 410may incorporate conductive heating and cooling, or radiant heating froman optical or infrared source for control of the temperature of thefluid samples, thereby providing the proper incubation.

In yet another embodiment of the instruments 10, 310, 410, the lightsource and sensor are fixed, and the multiple sample chambers are fixed.However, optical elements such as mirrors or prisms onelectro-mechanical actuators are used to move the light beam frommeasurement chamber to measurement chamber within each sample. Hence,the electro-mechanical actuators and possibly motors are used to movethe light beam, while the light source, the sensor(s), and the multiplesample chambers are fixed. In yet a further embodiment, there is a fixedsensor associated with each cuvette/sample position (e.g., such that theinstrument has 16 individual sensors) and only the light sourcetranslates.

In one mode of operation of the optical measuring system, the fluidsamples in the cuvettes or fluid chambers may be developed from a singlesample (e.g., from a single patient) constituted from one or multipleliquids and/or dry materials that are combined and mixed. Each of thefluid chambers could be pre-loaded with a chemoeffector including adrug, antimicrobial agent, nutrient, chemical tag or colorant. Eachmeasurement chamber is then sequentially measured with one or moreoptical beam lines, or by moving the beam lines around the sampleassembly. If each individual measurement chamber includes a differentchemoeffector (e.g., different dosage of an antibiotic), then the effectof the separate chemoeffector can be monitored over time for a singlefluid sample. Thus, each of the optical measurement instruments in FIGS.1-10 can be used to determine the effects of a chemoeffector (a drug,antimicrobial agent, nutrient, chemical tag or colorant) on a singlesample if the cuvette (such as cuvettes assembly 110) is loaded with asample from a single patient, but the chambers includes differentchemoeffectors (or each cuvette assembly 110 is designed to test asingle chemoeffector four times for accuracy/repeatability). Thus, inthe instrument 10 of FIGS. 1-5, a single patient's sample could betested against multiple chemoffectors. The codes on the label 170 on thecuvette assembly 110 may identify which chemoeffector is being testedwithin the respective assembly 110.

Regarding the operation of the instrument 10, one sample of test datafrom each fluid sample can be developed and recorded locally in thememory 60 within about 10 seconds. The laser 20 beam is transmittedthrough the sample contained between two windows, and into the sensor22. The sensor 22 captures the scattered light across its surface andmeasures the distribution of light intensity as a forward scattersignal, which is them stored locally for a period of time, before beingdownloaded (on a periodic basis) to a larger memory device (e.g., theMeasurements Database 620 in the network 600 in FIG. 19) that is linkedto the instrument 10. Similarly, the intensity of the laser beam on thesensor 22 can be measured in a location where there is no samplepresent, and again measured through the sample to determine the amountof power reduction that is attributable to absorption or reflectance ofthe enclosed sample, and the difference in these two values can be usedto calculate optical density for the sample. As such, the instrument 10can measure optical density of the fluid samples, which provides anotherpiece of data that can be used for determining the bacterialconcentration and its growth over a period of time. The optical bench 18then translates to the position corresponding to the next sample.Accordingly, if sixteen samples are present (4 cuvette assemblies 110,each with 4 sample chambers), then the all sixteen samples can becompleted in approximately 2-3 minutes. As such, the laser 20 and thesensor 22 continuously cycle through the fluid samples and measure aforward-scatter data point for each of the sixteen samples in about 2-3minutes. For example, in a 2-hour test period, twenty or more multiplescatter signals for each of fluid samples can be taken.

The instrument 10 measures bacteria and other organisms generally in therange for 0.1 to 10 microns with a measurement repeatability of 10%. Theinstrument 10 can measure a low concentration of 1×10⁴ cfu/ml (based onE-coli in filtered saline) and deliver continuous measurements showinggrowth beyond 1×10⁹ cfu/ml. The instrument 10 can be loaded withfactory-set calibration factors for approximate quantification of commonorganisms. Further, the user can load custom calibration factors withspecific test protocols for use with less common organisms or processes.

Considering that the particles in the fluid (especially bacteria) may bein in motion, it is possible that large clusters may affect theforward-scatter signal on any given test sample. Accordingly, in onepreferred embodiment, multiple consecutive test data points for eachfluid sample are averaged to avoid having a single forward-scattersignal with a large cluster of particles or a single forward-scattersignal corresponding to only a few particles affect the overall testresults. In one example, five consecutive forward-scatter signal testdata points are averaged under a rolling-average method to develop asingle average signal. Thus, as a new data point is taken for eachsample, it is used with the previous four data points to develop a newaverage. More or less data points than five can be used for this rollingaverage. Further, the computation methodology may use various algorithmsto remove the high and low signals (or certain ultra-high or ultra-lowsignals) before taking the average. Or, the computation methodology canbe as simple as choosing the mathematical median of a data set.Ultimately, the forward-scatter signals from the instrument 10 willproduce a bacterial-growth curve having a certain slope over a period oftime at an appropriate incubation temperature.

Generally, growth curves are numerically filtered and analyzed fordetermination of initial concentration, growth percentage for apredefined period of time, and changes in the growth rate. Determinationof bacterial absence or bacterial presence above a predefined thresholdis based on a combination of those parameters with thresholds that arecharacteristic for bacterial growth and salts crystallization/dissolvingkinetics. In one basic example, if the slope is above a predeterminedvalue, the patient's sample is infected. Alternatively, it could be thatthe slope that indicates the presence of an infection may be differentfor different periods of time (e.g., Slope_(infection)>X within T=0 to30 minutes; Slope_(infection)>1.5X within T=30 to 60 minutes; etc.)

Particles with a refractive index different from the surrounding mediumwill scatter light, and the resultant scattering intensity/angulardistribution depends on the particle size, refractive index and shape.In situations in which the input light is scattered more than one timebefore exiting the sample (known as multiple scattering), the scatteringalso depends on the concentration of particles. Typically, bacteria havea refractive index close to that of water, indicating they arerelatively transparent and scatter a small fraction of the incidentbeam, predominantly in the forward direction. With the optical designwithin the instrument 10, it is possible to look at scattering anglesdown to about 2° without having the incident input beam or other noisesignals (e.g., the scattering from the cuvette windows) interfere withlight scattered by bacteria. By simultaneous measurement of the forwardscattering and optical density, measurements could be extended down to10⁻⁵, allowing accurate measurement of concentrations as low as 10³CFU/mL.

Optical density measurements are intended to determine sampleconcentrations that are not accurate, as the size of the scatteringparticles greatly affects the resulting optical density. A similaroptical density is obtained for samples with a few large size bacteriain comparison with a higher concentration of small size bacterialsamples. Moreover, additional calibration of the optical density toconcentration does not render more accurate results, since the sizechanges during the bacterial growth process.

Bu use of the Mie scattering model for spherical particles and theT-matrix method of light scattering, combined with Monte-Carlo raytracing calculation that takes into account multiple scattering, it ispossible to evaluate the number of bacteria and their size from themeasurement of the optical density and the scattered light angulardistribution.

The results are nearly independent of the specific particle shape andloosely depend on the size dispersion of bacteria, resulting in a smallconstant shift of the mean size. Thus, both bacterial concentration andsize are evaluated from the measured parameters by a first principlemodel without any free parameters, except the bacteria refractive index,that is measured by calibration for each of the bacteria species. Inshort, the instrument 10 can be used to detect forward scatter signalscorresponding to scattering intensity and angular distribution (e.g.,for angles less than 5°, such as angles down to about 2°) and also theoptical density of the fluid samples, which can then be evaluated todetermine the number of bacteria and their sizes (and changes to thenumber of bacteria and to their sizes over a period of time).

FIG. 11 illustrates a screen shot 450 from the display device that wouldbe coupled to the instrument 10 via the USB communication port 30. Thebacterial growth curves for seven different patients' urine samples451-457 are illustrated. The curves are based on both forward-laserscattering and optical density testing, and correlated to estimatedorganism concentrations as discussed above. The samples 451, 456, and457 show bacterial growth, which is an indication of infection. Thesample 456 has only a slight slope, but enough to indicate that theparticles (bacteria) are increasing. On the other hand, the samples 451and 457 have much steeper slopes. On the other hand, samples 452 and 453have no substantially slope, which suggests no bacterial growth. Thesample 454 shows a little potential growth through its slight slope. Butbecause the sample 454 started high (suggesting a highly turbid sample),there may be a recommendation for further testing as there may be somebacterial growth hidden in the cloudiness. The sample 455 is a “rapidriser,” which is a sample with high concentration of phosphate saltsthat precipitate out in a cloud when warmed up, which causes the verysharp rise in particle concentration. This sample 455 represents a rareoccurrence (e.g., <5% of all cases) in testing using the device 10, andwould require further testing. In short, the patients corresponding tosamples 451, 456, and 457 have a clear infection, that can be identifiedand treated within 2-3 hours of testing. For a sample like the patientsample 451, the system can set off an audio and/or visual alarm (e.g.,blinking sample, flashing notification, scrolling banner across thebottom of the screen) once the slope reaches a certain level, indicatinga very high probability of an infection. (e.g., after 30 or 40 minutes)

FIG. 11 also shows raw-data region 460, which allows the operator toreview the actual test data for any given sample. Keep in mind that thescreen shot 450 for the display device is presenting information to theoperator who is monitoring five or ten instruments 10 that are inoperation. Thus, the operator can access the raw data for various testsand develop graphs of various samples by clicking on the raw datasamples within the raw-data region 460. And the graphs can be plotted inreal time on the display device.

Also shown on FIG. 11 are several possible features to facilitate use,including software icons for delivering various data screens to the userfor loading a new sample (shown as a “New Cuvette” button on FIG. 11,for archiving data on a separate data storage media (shown as a “datadisk” icon), for searching for particular sample results (shown as a“magnifying glass” icon), for pausing instrument operation (shown as a“play arrow” icon), and for opening the instrument door to load orunload samples (shown as a “Eject Arrow” icon).

Another icon shown in FIG. 11 is the “Biohazard” button, which isprogrammed to analyze a selection of samples to plot out organism growthas a function of the concentration of a particular chemoeffector orother value that has been linked to the samples. The screen on FIG. 11has other icons that can be used to control and monitor the instrument10, sort, analyze and visualize the loaded and collected data andresults, and operate the instrument 10. Other icons can be added ordeleted as the needs of the user are refined or evolve.

FIG. 12 illustrates a screen shot 470 from the display device that wouldbe coupled to the instrument 10 via the USB communication port 30. Thebacterial growth curves for eight samples 471-478 derived from the samebacteria-infected sample is illustrated, which in this case is used foranti-microbial susceptibility testing (AST). The difference in thesamples is the different levels of the antibiotics that have beenapplied to the sample. From sample 478, no RX treatment of the staphinfection has caused a large increase in the bacterial population. Andeven for the lower levels of the antibiotics shown in lines 475, 476,477, there is still some bacterial growth. However, once the antibioticlevel reaches 0.125 μg/ml, the antibiotic has killed the bacterialcolony as shown in sample line 474. Additionally, higher antibioticlevels, as shown in samples 471-473 yields no greater effect that sampleline 474. Accordingly, the instrument 10 can be used to determine thecorrect concentration of a certain antibiotic that should effectivelyeradicate a bacterial colony. It should be noted that the time scale onthe graph on the screen-shot 470 illustrates that the instrument 10 canprovide automated measurements for hours or days.

Like FIG. 11, FIG. 12 also shows a raw data field 480 for the AST thatidentifies the antibiotics being tested along with the bacteria types.The operator can scroll through the data and locate tests that he or shewants to review or have graphed.

The system and method associated with FIGS. 1-12 have various uses andapplications. For example, in the area of research, it can be used for(i) microbial concentration and grow analyses, (ii) quantification ofantimicrobial, antibiotics, and environmental effects, and (iii)antibiotic drug development and clinical trial enrollment. In the areaof hygiene and safety, it can be used for (iv) antimicrobial andantibiotics quality assurance testing, (v) process and potable watertesting, and (vi) surface, wipe, and swab microbial testing. In the areaof clinical microbiology for humans and animals, it can be used for(vii) rapid detection and quantification of infection, (viii) rapidantibiotic susceptibility testing (AST), (ix) drug-testing andmeasurement, and (x) antibiotic sensitivity testing for quality control.

The present invention associated with FIGS. 1-12 also contemplates theidentification (or partial identification) of the type of bacteria thatis present in fluid sample. For example, if a certain type of fluid isknown to have a limited number of types of bacteria, one type ofbacteria may be known to grow at a fast rate at a certain incubationtemperature relative to the other bacteria, leading to a higher slope onthe growth curves. One type of bacteria may be known to grow at a slowerrate at a certain incubation temperature relative to the other bacteria,leading to a lower slope on the growth curves. Or a group of bacteriamay be known to have certain growth curves, leading to the partialidentification by eliminating the other types of bacteria that may bepossibly present in the fluid sample. Using multiple instruments 10 withthe same set of fluid samples but at different incubation temperatures(e.g., the same sixteen samples in three instruments 10 at 38° C., 40°C., and 42° C.) can result in different bacterial-growth curves, whichidentify one type of bacteria relative another (or at least a species ofbacteria). Further, if one bacteria (or a species of bacteria) are knownto die above a certain temperature, then after the samples have beentested, the instrument 10 can ramp-up the temperature to see if thegrowth curve flattens for any sample, indicating that the sample may beinfected by the bacteria that is known to die above the operatingtemperature.

In a further example, complex UTI cases in humans are known to have bothGram Positive bacteria and Gram Negative bacteria. Crystal Violet is adye that adheres to the rough surface of Gram Positive bacteria and, inthe process, causes the pores on the surface to become “clogged” so asto kill the Gram Positive bacteria. Therefore, inclusion of CrystalViolet in one chamber of the cuvette assembly 110 while other chambersin the cuvette assembly 110 lack it permits identification of the UTIinfection type. If the bacteria growth curve continues similarly in bothchambers, then the patient's sample is likely infected by only a GramNegative bacteria. On the other hand, if the bacteria growth curve inthe chamber having Crystal Violet has a substantially smaller slope,then the infection likely includes a Gram Positive bacteria. As such, atleast a partial identification of the bacteria has been achieved. Inthis case, the chemoeffector is an inert chemistry (Crystal Violet) thatimpacts the growth behavior of the organisms, and by comparison to acontrol, some identification information for the bacteria can beobtained.

Regarding the identification of the bacteria, FIGS. 13 and 14 illustrateanother aspect of the present invention using additional devices andmethods to identify which type of bacteria is present within the fluidsample. In this aspect of the invention, as shown schematically in FIG.13, the optical-sensing instrument 10 is first used. Next, the systemmay involve the actions of a centrifuge 502 on a fluid sample from theoptical-sensing instrument 10. And finally, a mass spectrometer 504 isused on the fluid sample.

In FIG. 14, the process using the devices of FIG. 13 on a fluid sample(for example, urine samples) is illustrated. Initially, at step 510, thefluid samples from a variety of patients are placed into an incubatingand optical sensing instrument, such as the instrument 10 shown inFIG. 1. For example, fluid samples from sixteen patients can be placedin the four cuvettes 110 of FIG. 2, which are then placed within theincubating optical sensing instrument 10 in FIG. 1. If one or more ofthe sixteen fluid samples is detected as having bacteria, the incubationprocess continues until a desired concentration of bacteria is presentwithin the fluid sample (for example, a concentration of 1×10⁶ cfu/ml).In response to the forward-scatter signals for the sample as measured bythe device of FIG. 1 indicating the desired concentration, thecorresponding cuvette with the target fluid sample is then removed fromthe incubating and optical sensing instrument 10, and the fluid samplewith the desired concentration of bacteria is then removed from thecorresponding chamber of the cuvette by, for example, a pipette. At step512, the fluid sample with the desired concentration of bacteria fromthe cuvette is then placed into a centrifuge device to furtherconcentrate the bacteria into a smaller volume (e.g., 1×10⁹ cfu/ml),which permits the type of bacteria to be identified through a massspectrometer. A washing process (perhaps repeated) with purified watermay be used in conjunction with the centrifuging process to betterconcentrate the bacteria. After the centrifuging process, at step 514,the concentrated bacteria are placed into a mass-spectrometry microbialidentification device that can be used to identify the type of bacteriathat was within the original fluid sample (for example, a urine samplefrom a particular patient). This third device may be a Biotyper MatrixAssisted Laser Desorption Ionization-Time of Flight Mass Spectrometer(MALDI-TOF) from the Bruker Corporation, or a Vitek® MS device frombioMérieux SA. Other known devices that use a mass spectrometer toidentify the type of bacteria can be used as well. Accordingly, theoptical sensing instrument 10 is not only used to identify the presenceof bacteria, but it is then used to incubate the bacteria to achieve acertain concentration of bacteria that can then be used to conduct abacteria-identification process.

FIG. 15 provides a more detailed process flow for identifying the typeof bacteria within a plurality of different fluid samples using thecuvettes and incubating and optical measurement device 10. Inparticular, the fluid samples are loaded into the cuvettes 110 at step520. The cuvettes 110 are then loaded into the optical sensinginstrument, such as instrument 10, in step 522. The optical sensinginstrument determines if any of the fluid samples have bacteria presentat step 524. If bacteria are detected, the incubation continues for thepurpose of increasing the bacteria concentration to a predeterminedlevel at step 526. After the desired bacteria concentration is achieved,the cuvettes 110 are removed from the instrument 10 at step 528. Thefluid sample(s) having the desired bacteria concentration is achieved isremoved from the cuvette 110 (possibly by use of a pipette) at step 530,and the fluid is centrifuged at step 532 to achieve a fluid with ahigher bacteria concentration that is needed for mass spectroscopy. Itshould be noted that, in some instances, the centrifuge may not beneeded as the bacteria concentration from the prolonged incubationshould be high enough for identification via mass spectroscopy. Finally,at step 534, the fluid with the higher bacteria concentration from thecentrifuge is placed in a mass spectrometer and the bacteria is thenidentified.

Example 1

The following information provides one exemplary test process inaccordance to FIGS. 13-15, which is related to the use of e.coli((ATCC25922):

-   -   A freshly grown e.coli (ATCC25922) colony is diluted and placed        in a Luria Broth (LB) to achieve a 1×10⁴ CFU/ml concentration        level. The 1×10⁴ CFU/ml concentration level is the minimum        bacteria concentration for indicating a urinary tract infection.    -   Three ˜2 mL aliquots are loaded into three chambers of 4-chamber        multi-cuvette (FIG. 2), and the fourth chamber was used for LB        control to confirm sterility.    -   Concentration curves are obtained on ˜2 minute intervals in a        BacterioScan 216R instrument (FIG. 1), with samples heated to        37° C.    -   When the sample concentration reaches 10⁵, 10⁶, 10⁷ & 10⁸ cfu/ml        as detected by the BacterioScan 216R, (i) an aliquot of 1 μl is        loaded onto MALDI-TOF device (“wet target”) and (ii) an aliquot        of 1 ml is processed via a centrifuging/washing process into a        pellet (“dry target”) having a higher bacteria concentration,        which is then loaded into a MALDI-TOF device.    -   To confirm the sample concentrations, a 10 uL streak culture is        plated on 5% defibrinated sheep blood agar plates, incubated,        and colonies are counted.    -   A static culture (e.coli at 1×10⁴ CFU/ml in 25 mL of LB) is        created and incubated at 37° C., and processed as above (dry &        wet target for MALDI, with 10 ul plated on blood agar).    -   The entire experiment is performed twice—Day 1 & Day 2.    -   Day 1 dry samples are centrifuged for 2 minutes each.    -   Day 2 dry samples are centrifuged for 5 minutes each.

Regarding the results, only the “Dry target” method was successful inidentifying the type of bacteria via the MALDI. The “Wet target” methoddid not achieve a high enough concentration of bacteria before beingplaced in the MALDI to permit identification of the bacteria. However,the present invention contemplates that the “Wet target” method may workfor some types of bacteria and in certain fluids that will permit thebacterial concentration to substantially increase in the fluid sampleincubated within the BacterioScan 216R device over a longer period oftime (e.g., 8 hours).

FIGS. 16A and 16B illustrate the results from Day 1. In particular, FIG.16A illustrates the fact that the bacteria concentrations measured bythe BacterioScan 216R device (“B scan” from FIG. 1) were relativelyconsistent with the bacteria concentrations on the blood agar plates andthe static culture plates. Accordingly, the bacteria concentrationsmeasured by the BacterioScan 216R device are accurate for the purposesof the testing in Day 1. The concentration data for blood agar platesand static culture plates after 150 minutes are incomplete due tohigh/TNTC colonies. Each data point on FIG. 16A represents the time andconcentration levels at which the fluid samples were processed andloaded onto MALDI.

FIG. 16B illustrates the MALDI raw data for the “dry target” samples atcertain bacteria concentrations measured by the BacterioScan 216Rdevice. For the first two data points, which correspond to bacteria atthe 1×10⁴ CFU/ml concentration level and the 1×10⁵ CFU/ml concentrationlevel, the results were not reliable for determining for genusidentification or species identification. For the third data point,which corresponds to bacteria at the 1×10⁶ CFU/ml concentration level,the results allowed the genus identification to be secured, but thebacterial species identification was only probable. For the fourth andfifth data points, which correspond to bacteria at the 1×10⁷ CFU/mlconcentration level and the 1×10⁸ CFU/ml concentration level, theresults were reliable for determining the bacterial genus identificationand the species identification was highly probable.

FIGS. 17A and 17B illustrate the results from Day 2. In particular, FIG.17A illustrates the fact that the bacteria concentrations measured bythe BacterioScan 216R device (FIG. 1) were relatively consistent withthe bacteria concentrations on the blood agar plates and the staticculture plates. Accordingly, the bacteria concentrations measured by theBacterioScan 216R device are accurate for the purposes of the testing inDay 2.

FIG. 17B illustrates the MALDI raw data for the “dry target” samples atcertain bacteria concentrations measured by the BacterioScan 216Rdevice. The results were consistent with Day 1 testing. For the firsttwo data points, which correspond to bacteria at the 1×10⁴ CFU/mlconcentration level and the 1×10⁵ CFU/ml concentration level, theresults were not reliable for determining for genus identification orspecies identification. For the third data point, which corresponds tobacteria at the 1×10⁶ CFU/ml concentration level, the results allowedthe genus identification to be secured, but the bacterial speciesidentification was only probable. For the fourth and fifth data points,which correspond to bacteria at the 1×10⁷ CFU/ml concentration level andthe 1×10⁸ CFU/ml concentration level, the results were reliable fordetermining the bacterial genus identification and the speciesidentification was highly probable.

As can be seen by the graphs of the MALDI raw data in FIGS. 16B and 17B,when the bacteria concentration (as measured by instrument 10, theBacterioScan 216R instrument) reaches 1×10⁶ cfu/ml and the subsequentwashing/centrifuging process is used on the liquid samples to achievethe “dry target” pellets, the MALDI device was able to identify the typeof bacteria. In other words, at T=0, the concentration of the e.colibacteria was initially 1×10⁴ cfu/ml. Through the incubation processassociated with the instrument 10 (BacterioScan 216R) the growth ofbacteria over approximately 200 minutes caused the concentration of thebacteria to reach 1×10⁶ cfu/ml, which was enough concentration ofbacteria to result in the subsequently derived “dry target” samples topermit a reliable identification of the bacteria through the MALDIdevice. When the bacterial concentration went beyond the 1×10⁶ cfu/mllevel, the probability of identifying the bacteria was even greater. Assuch, the forward-scatter signal measurement associated with theinstrument 10 (BacterioScan 216R device) provides periodic growthinformation to determine the concentration of the bacteria over time,allowing for the detection of a predetermined bacterial concentration.Again, it should be noted that a bacteria concentration of approximately1×10⁴ cfu/ml is considered the minimum bacterial concentration for aurinary tract infection, and most human samples that have a urinaryinfection are higher than the 1×10⁴ cfu/ml bacterial concentration.

FIG. 18 illustrates the percentage of patients that have tested positivefor urinary tract infections, which has been identified as having athreshold bacteria concentration of 1×10⁴ cfu/ml. Data were collectedover several months from over 1500 UTI specimens using the BacterioScan216R device (FIG. 1). The data from the urine specimens was generallymeasured and gathered for 180 minutes, such that the graph lines between180 minutes to 300 minutes in FIG. 18 are extrapolations. As can beenseen from the upper curve (“UTI Detection” which is a 10⁴ cfu/mlconcentration), as the incubation period increases, the number ofpatients that are identified as having a urinary tract infection alsoincreases as the bacteria grows within the cuvettes stationed within theBacterioScan 216R device.

In FIG. 18, the bottom set of data corresponds to a bacterialconcentration of 1×10⁶ cfu/ml, which has been identified as the minimumamount of bacteria concentration that will permit bacteriaidentification through the “dry target” process using the MALDI deviceof Example 1 above, as shown in FIGS. 16-17. Focusing solely on thebottom set of data (1×10⁶ cfu/ml) of FIG. 18, within 60 min. of thetest, approximately 65% of the patients who have a urinary tractinfection have been identified and their bacteria concentration is atleast 1×10⁶ cfu/ml. Again, it should be noted that most human urinesamples that are infected have a bacteria level that far exceeds the1×10⁴ cfu/ml, which corresponds to the top curve (“UTI Detection”).Accordingly, at T=60 minutes, the present invention contemplates theincubation and optical measurement instrument of FIG. 1 will report backto the user (e.g., through the display device 14 of the instrument 10 oron a computer display linked to the instrument 10) a listing of thefluid samples that identify the approximately 65% of the fluid samplesthat can now be tested via the MALDI device because they have asufficient bacteria concentration. Other samples continue to residewithin the incubation and optical measurement instrument 10, until theoptical measurements associated with each of those samples indicates thebacteria concentration has finally achieved 1×10⁶ cfu/ml. In otherwords, by 120 minutes, another 5-6% of the fluid samples will have aconcentration level that is at least 1×10⁶ cfu/ml, such thatapproximately 70% to 71% of all samples having a bacterial infectionhave been identified and can be further processed to identify thespecific bacteria through the MALDI device. And by 300 minutes,approximately 80% of all samples with a bacterial infection have enoughconcentration of bacteria to permit the identification of the specificbacteria through the MALDI device. It should be noted that as of 300minutes, the remaining 20% of patients' samples having a bacterialinfection are still identified, but the level of the bacteria has notachieved the level of 1×10⁶ cfu/ml so as to permit reliableidentification via a “dry target” process within the MALDI device. Assuch, those remaining 20% of the samples may need to be “plated” forhigh-concentration bacterial growth over a longer time (24-48 hours)before placement within the MALDI device. However, the benefits of thepresent invention are substantial in that, unlike the currentstate-of-the-art which may take several days to identify the type ofbacterial infection within the urine, 65% of the patients who have aurinary tract infection (i) can be determined as having a “positive” UTIthrough the use of the optical measurement device, and (ii) can haveidentified the exact bacteria that caused the infection (which leads toa more rapid intake of the appropriate antibiotic or treatment plan).The overall process (including the 60 minutes of incubation time withinthe device) to identify the exact bacteria causing an infection is lessthan 2 hours.

The middle set of data in FIG. 18 (Labeled “3×10⁵ cfu/ml”) represents aconcentration level that is believed to be sufficient to permit theidentification of the specific bacteria through the MALDI device by useof an enhanced washing and centrifuging process. The enhanced processmay include methods of filtration or selective binding elements that canbe used to concentrate one species of bacteria preferentially to anotherwithin a single liquid sample, or a highly specific lysing orantimicrobial chemoeffector, bacteriophage, or other natural orsynthesized genetic agent, which may destroy or suspend the growth of aparticular bacteria while leaving other unidentified bacteriaunaffected, thereby reducing the prevalence of any known contaminant orother known organism and increasing the relative concentration of anunidentified organism so as to improve the probability of accuratedetection by MALDO-TOF or other method. This will result in a higherconcentration of bacteria that is placed as a “dry target” in the MALDIdevice. As such, the present invention contemplates a process by which80-90% of the patients who have a urinary tract infection (i) can bedetermined as having a “positive” UTI through the use of the opticalmeasurement device, and (ii) can have identified the exact bacteria thatcaused the infection within 2-3 hours. As such, relative to presentpractice, the inventive process results in 80-90% of patients have a UTIbeing treated much more quickly with the appropriate antibiotic ortreatment plan.

There are a few additional noteworthy details of the system and processof FIGS. 11-18. First, the onboard incubation and growth monitoring ofthe instrument 10 detects low (T=0) infections and slow growingpathogens such that the detection can be after the initial laser scan,or shortly thereafter. And, the broth-dilution protocol reduces relativeconcentration of contaminants, chemical preservatives and/or residualantibiotics in the patient sample, and thereby reduces the lag time forthe sample to enter into logarithmic growth rates resulting in earlierdetection, as well as a measured growth rate that is more consistentthan in the case of undiluted sample with unknown antibiotics orpreservatives, providing additional information for deductiveidentification of the infective organism. Lastly, improvements in theextraction efficiency of the bacteria into the “dry target” pellets willspeed results.

FIG. 19 illustrates a network 600 of digitally connected components thatinclude one or more test instruments 10, 310, and 410 and massspectrometers 504 (which may be coupled to the instruments 10 inaccordance with FIGS. 13-18), test setups, and/or remote workstationsthat generate measurement data (collectively “Test Assets”). The digitalnetwork 600 further includes at least two independent databases—ameasurements database 620 of raw collected measurements from the TestAssets and an identifiers database 630 that correlates the private eventrecord (e.g., Accession Numbers) to a data record identifier, such as aSample ID. The network 600 further includes a report-generator softwaremodule 640 that generates an interpreted result by correlating data fromboth the measurements database 620 and the identifiers database 630. Thereport-generator software module 640 analyzes the raw data and, by useof the correlated information in the identifiers database 630, deliversa report to the user via the Laboratory Information Systems (“LIS”) 645,which includes user input devices and output devices, preferably in amanner such that it does not create or store a permanent record of thecorrelated information.

The report-generator software module 640 (or a separate sample-loadingsoftware program) may be used to collect an Event Record for storageinto the Identifiers Database 630. The Event Record is an entry storedin the Identifier Database 630 that includes the Accession Number (orother hospital or facility record information) and the Sample ID, whichas described in more detail below, includes the serial number andchamber number for the cuvette assembly 110 as indicated by a codedlabel 170 (FIG. 6). In common practice, the Accession Number is part ofthe hospital's or facility's patient record and is within the associatedLIS 645. The Event Record may also include other data about the loadingof the sample (e.g., time, date, loader, testing protocol). Thefollowing is an exemplary Event Record:

-   -   Date: Apr. 20, 2015    -   Time: 3:35:44 GMT    -   Hospital Accession Number: MB042015-033    -   Lab Operator: 056 (Jane Q. Biotech)    -   Sample ID: 023409823402934 (Cuvette Assembly serial #)-1        (chamber #)    -   Test Type: Urinary Tract Infection Diagnostic, 5 μm filter, no        preservative, no yeast        The Accession Number is typically recorded in the hospital's        patient health record. The Sample ID would not be part of the        hospital's patient health record. It should be noted that the        Event Record could be recorded by a manual process in which the        laboratory enters the appropriate data.

The Sample ID can be collected from the cuvette assembly 110 by use of acode reader (such as a barcode or QR-code reader) that reads the code170, or the operator can simply type in the data read by eye.Alternately, the Sample ID could be generated by a random numbergenerator and stamped onto a barcode label and stuck to the cuvetteassembly 110, which disconnects the cuvette assembly 110 and all of itsinformational markings from the hospital identifications so as tofurther assure that the instrument 10 can never have anypatient-identified information.

Within the network 600, the instruments 10 (and other Test Assets) onlytransmit raw measurement data to the measurements database 620. Theinterpretation of the measurement data from the instrument 10 and thereporting are conducted by the report-generator software module 640.This interpretation of the measurement data from the instrument 10 isaccomplished by collecting the raw data from the Measurements Database620, and then performing calculations and analysis using that data todetermine results for the patient (e.g., Patient has a urinary tractinfection, or Patient does not have a urinary tract infection). Theformulas, algorithms, and reporting formats that the report-generatorsoftware module 640 uses to conduct these analyses are established byAlgorithms and User-Preferences module 643, which may be stored in someother database. The Algorithms and User-Preferences module 643 ishelpful because different laboratories may have different thresholds forwhat is a positive result (infection) versus a negative result (noinfection) depending on the patient, the location (nursing home vs.surgical suite), or the loading protocol or notes (e.g., “this samplewas bloody and required multiple filtering steps”). Hence, theAlgorithms and User-Preferences module 643 provides the ability toselect from different analytical methods, and possibly to even look at aset of measurements under several different analyses methods atdifferent times. Because the network 600 does not rely on the instrument10 (or any Test Asset) to provide a final test result (i.e., theinstrument 10 only provides the raw measurement data that is stored inthe measurements database 620), the network 600 provides the option toanalyze the patient's sample multiple times (perhaps at points later intime than the initial test) under different analytical protocols becausethe report-generator software module 640 can retrieve different optionalsettings from the Algorithms and User-Preferences module 643. The LIS645 permits the user to provide inputs and view/retrieve outputs (on adisplay or in paper) by use of the report-generator software module 640.

As shown in FIG. 19, the report-generator software module 640 and theUser-Preferences module 643 can be stored locally within the laboratoryand updated as needed. Alternatively, report-generator software module640 and the Algorithms and User-Preferences module 643 are remotelylocated, such as in a remote cloud databases (like the separatedatabases 620, 630, and 650). The laboratory user then accesses thereport-generator software module 640 and hence Algorithms andUser-Preferences module 643 via a Java applet that is temporarilyrunning its Graphical User Interface (GUI) on the user's laboratorycomputer, while the software and settings are remotely stored andrunning. As such, the network 600 can be hosted remotely and isaccessible via the user's laboratory computer or tablet, via an app.

The network 600 may include a third database 650 (“The PrivateDatabase”) that contains additional patient or potentially privateinformation that is indexed to the Accession Number and protected as ifit is Patient Identifiable Information (PII). The report-generatorsoftware module 640 may access, retrieve, and use this information inthe process of analysis for interpretation, or for assembling a reportthat is specific to the patient. The report-generator software module640 may generate or modify this third database 650 with interpretedresults, raw data, or a record of an interpretation event or generationof a report (e.g., an event log).

A data-mining software module 660 is used to search or aggregate largeamounts of raw test data from the measurements database 620, includingthe test type, the measured results, the date of test, and/or theidentity of the Test Asset (e.g., the instrument 10). The data-miningsoftware module 660 may analyze the data for improving the quality orutility of the collected data, or for improving future use of thenetwork 600 or data within the network 600 for purposes such as publichealth surveillance, or for other purposes. Within the network 600, thisdata-mining software module 660 would not access any of the data fromthe Identifier Database 630 or the Private Database 650. Therefore, thedata used and the results generated by the data-mining software module660 would be devoid of any private information or information that couldbe combined or construed to be private information. As such, the network600 would permit data analysis without the burdens placed to protectpotentially private information.

One exemplary use of the network 600 will be described relative to FIGS.19-21 with reference to the instrument 10 and the cuvette assemblies 110described in FIGS. 1-8. A first patient is suspected of having a urinarytract bacterial infection and a fluid sample from the first patient isdelivered to the laboratory for testing. With reference to FIG. 20, thelaboratory technician receiving the fluid sample for the first patientis instructed to select a certain test kit from an inventory of testkits 700 that is to be used for the suspected urinary tract infection.In this case, the laboratory technician is instructed to select Test Kit#10 from an inventory 700. Test Kit #10 includes four cuvettesassemblies 110 a, 110 b, 110 c, and 110 d as shown in FIG. 21. Each ofthe four cuvettes assemblies 110 a, 110 b, 110 c, and 110 d includes apreloaded type and amount of chemoeffectors (e.g., antibiotics) thatcould be used for treating the suspected type of urinary tract infectionin the first patient. One of the assemblies 110 may include one or morecontrol chambers that lack a chemoeffector. The laboratory technicianthen loads (perhaps after a filtering procedure) each of the sixteenchambers within the four cuvettes assemblies 110 a, 110 b, 110 c, and110 d with the fluid sample from the patient.

Each of the Test Kits #1-12 in FIG. 20 includes one or more cuvetteassemblies 110 and is directed to a certain testing protocol. One ormore chemoeffectors are present, and/or the same chemoeffectors may bepresent at different concentrations in the various chambers (as shown inthe chemoeffector results of FIG. 12). In one preferred embodiment, anexperimental chemoeffector (e.g., experimental drug) may be present inthe one of the cuvette assemblies 110 of a Test Kit so that it can betested against commonly used drugs. Further, one cuvette assembly 110(or one or two chambers of one cuvette assembly) may include a chamberas a control for the test, which includes no chemoeffectors.

Regarding the identification of the cuvette assemblies 110, each of thecuvettes assemblies 110 a, 110 b, 110 c, and 110 d preferably includesthe coded label 170 (FIG. 6) that can be scanned by the laboratorytechnician prior to placement in the instrument 10 via the door 12(FIG. 1) or can be read via an internal scanning/reading device withinthe instrument 10. The code or code(s) on the coded label 170 correspondto the testing used for Test Kit #10, which is focused on the suspectedurinary tract infection. A first type of information from the codedlabel 170 identifies the specific cuvette assembly 110 and its chamberthat is being used, which together form the “Sample ID” show in FIG. 19.The Sample ID may also include the chemoeffector content within thechamber. A second type of information from the coded label 170 instructsthe instrument 10 regarding the test protocol, such as the incubationtemperature, test duration, and time intervals between taking test databy use of the laser 20 and the sensor 22 of the instrument 10. Thelaboratory technician also enters the Accession Number associated withthe first patient via the LIS 645. The Accession Number is indexed tothe Sample ID (e.g., cuvette assembly serial number and chamber number),which together comprise the Event Record. The software module 640 thensends the Accession Number and corresponding Sample ID to theidentifiers database 630 for storage as an Event Record. To the extent aseparate private database 650 is used as part of the network 600, thenetwork 600 may store the Accession Number along with other privateinformation related to the first patient.

The instrument 10 then performs the testing on the first patient'sliquid sample within the cuvette assemblies 110 a, 110 b, 110 c, and 110d. The raw data measurements for the testing from the instrument 10,including bacterial concentration data (e.g., bacterial concentrationcurves) over a period of time, are then stored within the measurementsdatabase 620. It is noteworthy that the measurements database 620 lacksany personal information regarding the patient. Rather, it includesinformation regarding the type of cuvette assemblies 110 a, 110 b, 110c, and 110 d that have been tested, the chemoeffectors (e.g.,antibiotic) contents of the cuvettes, and the raw data from the testingwithin the instrument 10. Considering that the measurements database 620is storing information from multiple remote laboratories in which theinstruments 10 are being used, the measurements database 620 contains anabundance of important biological information and data that can beanalyzed and reported through the data-mining software module 660 to ananalytics user.

Meanwhile, after a test has been completed, the laboratory technicianusing the LIS 645 can access both the measurements database 620 and theidentifiers database 630 by use of the report generator software module640 (and the algorithms and user preferences module 643) to develop areport specific to the first patient whose fluid sample has been tested.The report can then be sent back to the hospital and/or doctors treatingthe first patient that indicates the results. The results can bepresented in various forms such as, (i) the first patient has or doesnot have a urinary tract infection, (ii) the first patient has a urinarytract infection treatable by antibiotic X, (iii) the first patient has aurinary tract infection treatable by antibiotic X or antibiotic Y, (iv)the first patient has a urinary tract infection treatable by a firstpredetermined concentration of antibiotic X, and/or (v) the firstpatient has a urinary tract infection treatable by a first predeterminedconcentration of antibiotic X or a second predetermined concentration ofantibiotic Y. The report can be developed and/or reported manually orautomatically through the LIS 645 associated with the laboratory.

By use of the data mining software module 660, the network 600 providesaccess to non-private data derived from the instruments 10 (and otherTest Assets) within the measurements database 620 that can be used fornumerous functions related to determining and/or predicting the effectsand results of various chemoeffectors, such as:

-   -   Direct comparison of multiple antibiotics against a certain        infection on fluid samples from a large population of patients    -   Direct comparison of the same antibiotic at different        concentrations against a certain infection on fluid samples from        a large population of patients    -   Direct comparison of a new drug against known drugs on fluid        samples from a large population of patients    -   Detection of the emergence of one or more incidents of resistant        infection in any healthcare site or geographic region at any        time    -   Determination that a certain type of bacteria has become or may        be becoming (i.e., a prediction) resistant to a certain        antibiotic    -   Determination that a certain type of bacteria in a certain        geographical region has become or may be becoming (i.e., a        prediction) resistant to a certain antibiotic    -   Determination that a certain type of bacteria in a certain        hospital or care unit has become or may be becoming (i.e., a        prediction) resistant to a certain antibiotic    -   Determination of the susceptibility or resistance of an        infection pathogen to an antimicrobial agent, molecule, or        combination or sequence of exposure of antimicrobial agent or        molecule with or without the active involvement of the proximate        healthcare providers or clinical microbiologist

The data mining software module 660 is stored within a memory devicewithin or accessible by a computing system 670 having various hardwarecomponents (e.g., processors) and/or software or firmware components,modules, or features. The computing system 670 may include a smartphone,a laptop, a tablet computing device, a personal computer, or the like.The computing system 670 can be connected to the measurements database620 through a public or private network, such as the Internet. Thecomputing system 670 includes one or more input devices for receivinginputs from the analytics user, and one or more display devices fordisplaying outputs to the analytics user.

The analytics user that accesses the data within the measurementsdatabase 620 via the data-mining software module 660 can input variousqueries to determine and predict trends by use of the raw test datawithin the measurements database 620. In particular, when the instrument10 and the associated cuvettes assemblies 110 are tested as describedabove, the raw data includes the concentration of bacteria in the liquidsample over a period of time. As the bacteria grow during the incubationperiod, the concentration (i.e., the number of bacteria “particles”)increases, resulting in a different forward-scatter signal. As such,this test data can be in the form of graphical curves of bacterialconcentration versus time. Accordingly, the analytics user may include aquery related to locating a certain slope of the curve at a certainpoint in time. For example, after two hours, if the slope of the curvebegins to approach a horizontal asymptote, such that the slope isapproaching zero, then the growth of the bacteria within the liquidsample has subsided. In that scenario, identifying the chemoeffector(s)and/or the concentration of the chemoeffector(s) that prohibited orinhibited bacterial growth would be predictive of future treatments forpatients having a similar condition. Accordingly, the analytics user mayinput queries into the data-mining software module 660 to locate SampleIDs with raw data results in which (i) the bacterial growth curve has acertain slope during an initial period of time, (ii) the bacterialgrowth curve has a certain slope after a certain period of time afterwhich the chemoeffector(s) has begun to inhibit the bacterial growth,(iii) the initial bacteria concentration is a certain level, (iv) thebacterial growth curve for the “control” test for that sample has acertain higher slope to indicate the presence of growing bacteria, (v)the samples are identified in which the difference between slope(s) forone or more chemoeffector(s) test(s) and the control test is above acertain threshold.

While the network 600 of FIG. 19 has been described in connection withthe use of the instrument 10 and the associated cuvette assemblies 110,other test instruments (such as the instruments 410) can be used aswell. As described above with reference to FIGS. 13-18, after bacteriais detected and confirmed in a control sample by one of the instruments10, 410, the sample fluid can undergo a centrifuging process, and theconcentrated bacteria is placed into a mass-spectrometry microbialidentification device 504 that can be used to identify the type ofbacteria that was within the original fluid sample (for example, a urinesample from a particular patient). As such, the Test Assets may includea mass spectrometer device 504, such as Biotyper Matrix Assisted LaserDesorption Ionization-Time of Flight Mass Spectrometer (MALDI-TOF) fromthe Bruker Corporation, or a Vitek® MS device from bioMérieux SA, suchas those discussed relative to FIGS. 13-18. Other known devices that usea mass spectrometer to identify the type of bacteria can be used aswell. The detected type of bacteria can also be sent to the measurementsdatabase 620 for storage such that queries from the data mining software660 can be based on bacteria type.

FIG. 22 illustrates a flowchart that illustrates one method that can beused with the network 600 of FIG. 19. At step 802, a laboratory receivesa fluid sample from a patient. At step 804, the fluid sample is placedin multiple cuvette assemblies 110 that can be used for testing, such ascuvette assemblies that can be used for optical forward-scatteringmeasurement. At step 806, each of the cuvette assemblies 110 that willundergo testing that is identified with the Sample ID. Next, at step808, the accession number associated with the hospital/patient and theSample IDs to be used with the patient's fluid sample are stored in afirst database. Next, the test is conducted on the multiple cuvetteassemblies 110 at step 810. At step 812, the raw data from the testingon the multiple cuvette assemblies 110 is recorded in a second databasethat is different from and separate from the first database. At step814, a report can be developed for the patient indicating the finalresult of the testing by accessing first database (which has the rawtest results) and the second database (which has the accession numberand the sample IDs). Finally, at step 816, because the second databasedoes not contain any patient data, the raw test data from the firstpatient can be grouped together with other raw test data from otherpatients and accessed by data mining software to develop variousanalytics report.

Accordingly, the present invention relates to a network for medicaldiagnostic testing data where data is stored in a manner that isinherently untainted by patient identifiable information or anycollection of data that might be construed to be private patientinformation. Data from instruments networked within such a system may betransmitted, stored, aggregated, analyzed, and re-interpreted withoutconcern about patient privacy or data security, reducing the burdens ofdatabase and network design, operation, maintenance and use

Additionally, it should be noted that the present invention contemplatesa physical library of a plurality of test kits (e.g., test kits in FIGS.20-21), wherein each test kit includes one or more cuvette assemblies110 preloaded with chemoeffectors (and perhaps a control) that aredesigned for use in certain test protocols for different liquid samplessuspected of having different types of bacterial content. Each test kitmay include a code (e.g., the coded label 170 on the assembly 110) thatdictates the test protocol (e.g., duration, incubation temperature,forward-scatter sensing time intervals) to be used by the instrument 10.Once the code 170 is inputted into the instrument 10 and the cuvetteassemblies 110 are loaded into the instrument 10 via the door 12, thetest can begin in accordance with the protocol. The raw test data fromthe instrument(s) 10 for a large population of the patients is stored asinherently non-private information within the measurements database 620that is accessible to an analytics user via the computing system 670 toobtain important information regarding the determination of the resultsand/or the prediction of the effects of various chemoeffectors onbacteria.

Furthermore, the user may also be within a specific facility (e.g., ahospital) that accesses the measurements database 620 via the LIS 645and uses the data mining software 660 locally to determine the testresults on large samples of patients within that particular facility.Considering the benefits of the quick identification of bacterialinfections by the instrument 10 (relative to typical plating techniquesthat take 24 to 48 hours), the user at facility is more capable ofidentifying an infectious disease outbreak with that particularfacility. Consequently, the present invention contemplates a method ofloading a plurality of patient samples in the cuvette assemblies 110,using the instrument(s) 10 to gather the samples' test data that is thenstored in a database, and identifying, by accessing the database, atrend of bacterial infections within the particular facility. All ofthese steps can be performed in less than 24 hours, and oftentimeswithin 12 hours so as to avoid the need for the time-consuming “plating”steps. The steps may further include the process generally describedrelative to FIGS. 13-18 to identify the bacteria that is leading to theinfections. Ultimately, this process permits the facility to utilize theappropriate antibiotics to quickly limit the spread of the infectionwithin the facility.

Each of these embodiments and obvious variations thereof is contemplatedas falling within the spirit and scope of the claimed invention, whichis set forth in the following claims. Moreover, the present conceptsexpressly include any and all combinations and subcombinations of thepreceding elements and aspects.

1-19. (canceled)
 20. A network for collecting and using biological datarelated to pathogen within fluid samples, comprising: a plurality ofinstruments that are at remote locations, each of the plurality ofinstruments for testing a forward-scatter signal that is used todetermine the presence of a pathogen in a fluid sample from a patient; afirst database for storing a set of raw test data for each fluid samplefrom the plurality of instruments, each set of raw test data beingstored in a manner that is indexed to a test sample ID, the firstdatabase lacking any private patient information; a second database forstoring an event record that associates the test sample ID and a patientID; a report-generator software module that accesses information fromthe first database and the second database to develop a test report foreach patient; and a data-mining software module that accessesinformation from only the first database to determine or predict trendsfrom the raw test data.
 21. The network of claim 20, wherein theplurality of instruments determine a concentration of the pathogen inthe fluid samples.
 22. The network of claim 21, wherein the plurality ofinstruments assist with determining a type of pathogen in the fluidsamples.
 23. The network of claim 20, wherein the raw test data is inthe form of concentration of the pathogen over a period of time.
 24. Thenetwork of claim 20, wherein each fluid sample is placed in a pluralityof cuvettes.
 25. The network of claim 24, wherein the plurality ofcuvettes includes different chemoeffectors intended to effectconcentration of the pathogen.
 26. The network of claim 24, wherein theplurality of cuvettes include coded information that is a part of thetest sample ID.
 27. The network of claim 20, wherein the data-miningsoftware module accesses the information by queries related to a slopeof a curve defining concentration of the pathogen as a function of time.28. The network of claim 20, wherein the report-generator softwaremodule analyzes the raw test data from the first database to determine apatient's condition.
 29. The network of claim 20, further including athird database that is accessible by the report-generator softwaremodule, the third database including private patient information that isassociated with the patient ID.
 30. The network of claim 20, wherein thedata-mining software module provides an indication of or a detection ofan emergence of one or more incidents of infection from the pathogen inany healthcare site or geographic region.
 31. A network for collectingand using biological data related to pathogen within fluid samples,comprising: a plurality of instruments that are at remote locations,each of the plurality of instruments for testing a forward-scattersignal that is used to determine the presence of a pathogen in a fluidsample from a patient; a first database for storing a set of raw testdata for each fluid sample from the plurality of instruments, each setof raw test data being stored in a manner that lacks private patientinformation; and a data-mining software module that accesses informationfrom only the first database to determine or predict trends from the rawtest data related to at least one of the group consisting of: (i) anindication of or a detection of an emergence of one or more incidents ofinfection from the pathogen in any healthcare site or geographic region,(ii) an indication of or a detection of a certain type of pathogen in acertain geographical region has become or may be becoming resistant to acertain antibiotic, (iii) an indication of or a detection of a certaintype of pathogen in a certain hospital or care unit has become or may bebecoming resistant to a certain antibiotic, and (iv) an indication of ora detection of the susceptibility or resistance of the pathogen to anantimicrobial agent, molecule, or combination or sequence of exposure ofantimicrobial agent or molecule with or without the active involvementof the proximate healthcare providers or clinical microbiologist.
 32. Anoptical measurement system for use in detecting of a pathogen within afluid sample, comprising: a light source for producing an input beam; aremovable cuvette assembly for receiving the fluid sample, the cuvetteassembly having an input window for receiving the input beam and anoutput window, the cuvette assembly remaining in a fixed position withinthe system during operation; a sensor located outside the removablecuvette assembly for receiving a forward-scatter signal exiting from theoutput window, the forward-scatter signal being caused by the input beampassing through the fluid sample containing the pathogen; a heatingsystem that permits a controlled incubation temperature for the fluidsample; and a moveable optical bench undergoing translational movement,the light source and the sensor being mounted on the movable opticalbench, the translational movement of the optical bench permitting thefluid sample within the cuvette assembly to be placed into a path of theinput beam such that the sensor receives multiple forward-scattersignals for the fluid sample over a period of time.
 33. The opticalmeasuring instrument of claim 32, wherein the removable cuvette assemblyis placed on a registration platform.
 34. The optical measuringinstrument of claim 33, wherein the cuvette assembly is loaded through adoor on a housing of the optical measuring instrument and placed on theregistration platform.
 35. The optical measuring instrument of claim 33,wherein the heating system is located on the registration platform. 36.The optical measuring instrument of claim 32, wherein the cuvetteassembly has a plurality of internals walls defining a plurality ofindividual internal fluid containers within the single cuvette assembly,each of the fluid containers holding a corresponding one of theplurality of fluid samples.
 37. The optical measuring instrument ofclaim 32, further including a display device coupled to the opticalmeasuring instrument, the display device for displaying a growth curveof the pathogen for the fluid sample.
 38. The optical measuringinstrument of claim 37, further including an alarm for indicating when agrowth curve exceeds a certain slope or the forward-scatter signalexceeds a certain value.